The identification of tumor-associated antigens recognized by T-cells and a better understanding of how these T-cells are activated has renewed interest in the use of tumor vaccines for the treatment of cancer. The two signal hypothesis of T-cell activation states that T-cells require both an antigen-dependent signal delivered by HLA restricted epitopes and an antigen-independent signal delivered by co-stimulatory molecules. In fact, the lack of co-stimulatory molecule expression by tumor cells predicts that T-cells may be suppressed at sites of active tumor growth. This application seeks to understand the potential role of introducing co-stimulatory molecules into growing tumors as a method for enhancing local and systemic T-cell responses against the tumor. Recombinant poxviruses have been utilized to express human genes because of their stability, replication accuracy, and strong immunostimulant properties. The first aim is to study a recombinant vaccinia virus expressing the human B7.l co-stimulatory molecule in a dose escalation phase I trial in patients with malignant melanoma. The vaccine will be administered monthly as a direct intra-tumoral injection in an effort to activate tumor-infiltrating lymphocytes and evaluate the effects on systemic immunity. Patients will be evaluated for toxicity, clinical response, and systemic immune response by IFN-gamma ELISPOT assay. The second aim is to evaluate a novel recombinant vaccinia virus expressing three co-stimulatory molecules (B7.1, ICAM-1, and LFA-3), which has been superior to vaccinia-B7.l in pre-clinical studies. This vaccine will be tested in a similar dose escalation phase I clinical trial with similar endpoints. The third aim will be to evaluate the local effects of the vaccine through quantitative real-time PCR of fine needle aspirates taken from injected tumor lesions. Completion of these aims will demonstrate the safety and immunological effects of direct tumor injection of recombinant vaccinia viruses expressing co-stimulatory molecules. This may have important implications for the future design of tumor vaccines in melanoma and other settings.